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. 2018 Nov 21;25(1):13–21. doi: 10.1177/1753425918812646

Figure 2.

Figure 2.

Osteoclast differentiation induced by the three E. faecalis LTAs in osteoclast precursors was evaluated with the TRAP staining assay. (a and b) WT BMMs and (c and d) RbpjΔM/ΔM BMMs were treated for 6 d in the presence of 40 ng/ml M-CSF with the three E. faecalis LTAs, RANKL, E. faecalis LTAs plus RANKL, or pre-treated with RANKL for 3 d prior to treatment with E. faecalis LTAs for an additional 3 d, respectively. The cells were then subjected to TRAP staining. TRAP staining assay was carried out and visualized at 100× magnification under an inverted bright field microscope. Bar, 100 µm. The numbers of TRAP-positive multinucleated cells with more than 3 nuclei were counted from 6 random fields of view at 40× magnification. 1, Untreated cells; 2, RANKL (80 ng/ml); 3, RANKL (80 ng/ml) and E. faecalis ATCC 29212 LTA (50 µg/ml); 4, RANKL (80 ng/ml) and E. faecalis P25RC LTA (50 µg/ml); 5, RANKL (80 ng/ml) and E. faecalis P52Sa LTA (50 µg/ml); 6, Pre-treated with RANKL (20 ng/ml) and E. faecalis ATCC 29212 LTA (50 µg/ml); 7, Pre-treated with RANKL (20 ng/ml) and E. faecalis P25RC LTA (50 µg/ml); 8, Pre-treated with RANKL (20 ng/ml) and E. faecalis P52Sa LTA (50 µg/ml); 9, E. faecalis ATCC 29212 LTA (50 µg/ml); 10, E. faecalis P25RC LTA (50 µg/ml); 11, E. faecalis P52Sa LTA (50 µg/ml). The mean and SD are shown. *P < 0.05 was considered statistically significant compared with the untreated cells or RANKL-only treated cells. MNCs, multinucleated cells.